Research Challenge Area: Translational Studies 15 , Challenge Topic 04-HL-102 to Develop Integrated Strategies to Elucidate Mechanisms of Lung Disease Summary/Abstract Project Synopsis: This Challenge Research project will undertake 1) a mouse genome-wide shRNA library screen to identify genes and associated gene networks inducing or inhibiting goblet cell differentiation and mucous hypersecretion, and 2) an extensive small molecule compound library screen to identify compounds and processes regulating mucous cell activity relevant to both acute and chronic lung diseases. Mouse and human Respiratory Epithelial Cells (RECs) will be engineered with genes indicating the activity of a genetic network controlled by Spdef (Sam pointed domain Ets-like factor), a gene we recently showed is a "master regulator" of mucous cell programming in the lung. RECs will be engineered to express SPDEF and/or FOXA3 (using lentiviral vectors) and two distinct SPDEF target gene expression cassettes (Agr2-luciferase and Muc5A/C-renilla). A robotic, high-throughput screen will be undertaken to utilize a mouse shRNA library and a large, well-characterized chemical library screen human bronchial epithelial cells (HBECs) and mouse lung epithelial (MLE) cells to identify genes regulating SPDEF localization, levels, and target gene expression (activity). Genes and molecules inhibiting and those modulating SPDEF activity induces SPDEF nuclear localization and stability will be identified and characterized. The sites of expression of candidate genes and their association with the pulmonary disease processes complicated by mucous cell hyperplasia and mucous hyperproduction will be assessed in tissue from mouse models (in vivo) and in REC cells (in vitro), utilizing QRT-PCR, immunohistochemistry, and in situ hybridization. The nature of the genetic and chemical signaling pathways regulating goblet cell activity, will be modeled and candidate genes and molecules prioritized for further studies. The project takes advantage of recent identification of the important role of SPDEF in the pathogenesis of lung diseases associated with goblet cell hyperplasia and mucous hyperproduction. The accessibility of a near genome-wide lenti-shRNA library recently acquired by CCHMC and the outstanding high-throughput screening facility present at the Genome Research Institute (GRI) that was founded in partnership with the University of Cincinnati and CCHMC makes this large scale project feasible. The project will provide insights into the pathogenesis of goblet cell hyperplasia, mucous cell hyperproduction, create reagents for the study of pulmonary diseases affecting the airways, and identify genes and molecules of therapeutic interest for the treatment of common, chronic and acute pulmonary disorders, including CF, COPD and asthma. This proposal is responsive to the broad challenge area 15 "Translational Science" and Challenge topic 04-HL-102, and is based on our recent identification of the critical role that SPDEF plays in controlling a network of genes that determines goblet cell hyperplasia and mucous hyperproduction. Goblet cell hyperplasia and abnormalities of mucous cell secretion and function accompany and contribute to the pathogenesis of common pulmonary diseases, including asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). Likewise, mucous hyperproduction is associated with acute and chronic infection and inflammation that contributes to chronic upper respiratory disorders, including chronic sinusitis, allergic rhinitis, middle ear disease, as well as pulmonary disease that contribute to respiratory morbidity and mortality world wide. The identification and utilization of molecules that regulate goblet cell hyperplasia and mucous hyperproduction will be useful in understanding the pathogenesis of lung disease, and improving the diagnosis and treatment of common respiratory disorders. The present application is based on our preliminary data and the hypothesis: that goblet cell differentiation and mucous hyperproduction are mediated by signaling and transcriptional networks controlling Spdef activity regulate mucous cell activity in the respiratory tract. The application focuses to the identification of both genes and small molecules useful in controlling the expression and function of Sam pointed domain Ets-like factor (SPDEF). Goblet cell hyperplasia and mucous cell hyperproduction are associated with and contribute to the pathogenesis, morbidity and mortality attributed to common chronic and acute respiratory disorders, including Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis (CF), and asthma, together, lung diseases that affect millions of individuals world-wide. At present, there is little understanding of the genes, genetic networks, and precise pathological consequences related to goblet cell differentiation and a lack of markers and potential therapeutic targets to predict susceptibilities and improve outcomes, respectively. This project will identify genes and molecular pathways regulating goblet cell differentiation and mucous hyperproduction that will provide diagnostic and prognostic insight into chronic lung disease, and its impact on innate immunity and secondary infections commonly associated with these disorders. This project will identify potential therapeutic targets and molecules that will provide the basis for future therapies of these chronic lung diseases that, together, comprise a major health problem world-wide.